Plaint Physiol.

('1967") 42, 6-14

Ionic Balance in Different Tissues of the Tomato Plant in Relation to Nitrate, Urea, or Ammonium Nutrition
E. A. Kirkby and K. Mengel1
Department of Agricultural Chemistry, the University, Leeds, 2, England Received June 27, 1966.

Sumnwarv. An investigation was carried ouit to study the cation-anion balance in different tissues of tomato plants suipplied with nitrate, urea, or ammonium niitrogen in water culture. Irrespective of the form of nutrition, a very close balance was found in the tissues investigated (leaves, petioles, stems, and roots) between total cations (Ca, Mg, K and Na), and total anions (N03-, H2P04-, SO4--, Cl-) total non-volatile organic acids, oxalate, and uronic acids. In comparison with the tissues of the nitrate fed plants, the corresponding ammonium tissues contained lower concentrations of inorganic cations, and organic acids and a correspondingly higher proportion of inorganic anions. Tissues from the urea plants were intermediate between the other 2 treatments. These results were independent of concentration or dilution effects, caused by growth. In all tissuies approximately equivalent amounts of diffusible cations (Ca", \Ig+, K' and Nat), and diffusible anions (No3-, SO4--, H,PO4-, Cl-) and non-volatile organic acids were found. An almost 1:1 ratio occurred between the levels of bound calcium and magnesium, and oxalate and uronic acids. This points to the fact that in the tomato plant the indiffusible anions are mainly oxalate and pectate. Approximately equivalent values were found for the alkalinity of the ash, and organic anions (total organic acids including oxalate, and uronic acids). The influence of nitrate, urea, and ammonitum nitrogen nutrition on the cation-anion balance and the organic acid content of the plant has been considered and the effects of these different nitrogen forms on both the pH of the plant and the nutrient medium and its conseqtuences discussed.
Ionic equilibrium in plant tissues is maintained by diffusible and indiffusible ions, in which both organic and inorganic cations and( anions play a part. It is known that this equilibrium is very much dependent on the form of nitrogen nutrition to the plant (6, 9, 14). Previous studies have centered mainly on the comparison of nitrate and ammonium sources on the diffusible ionic balance in leaves, (6, 9,14) or oIn the influence of these nitrogen forms on the uptake of other ions (19). These experiments have shown that in comparison with ammonium fed plants, nitrate plants contain higher concentrations of cations and organic acid anions, while the content of inorganic anions is lower. Such observations have been explained by the axiomatic necessity for all plant tissues to maintain ionic equilibrium.
In order to investigate how different plant tissues maintain this equilibrium and the contributions of variouis components responsible for electroneuK. Mengel. Institut fuir Pflanzeilerinahrung der Justus Liebig IUniversitdit, Giessen, Germanv.

trality, both diffusible and indiffusible iOlns were determined in the leaves, petioles, stems, and roots of tomato plants supplied with nitrate, urea, or ammonium nitrogen. The inclusioni of urea as a comparative treatment also made it possible to substitute one main ionic component of the nutrient solution by an undissociated molecule. It was thus possible to compare systems in which the nutrient being taken tip by the plant in greatest quantities, nitrogen, was in the form of an anioin, molecule or cation. It is well known that the form of nitrogen nutrition influences the pH of the nutrieint solution (20). As this pH shift should be related to nitrogen metabolism, the present paper also considers the relationships between electroneutrality of the tissue, pH shifts in the medium andc aspects of nitrogein assimilation and metabolism.

Materials and Methods

Tomato plants (\var Ailsa Craig) N-ere groxwn from seed in the glasshouise in a soil comllpost mix-

KIRKBY AND MENGEL-IONIC BALANCE IN RELATION TO NITROGEN NUTRITION

ture. At the 4 leaf stage of growth the plants were removed from the soil, and by thoroughly rinsing in running tap water followed by distilled water all adhering soil particles were removed from the roots. The plants were then transferred to aerated nutrient solutions held in 7 liter black polythene containers, each container supporting 2 plants held by rubber foam in a 2 cm thick polystyrene sheet. The 3 treatments, nitrate, urea, and ammonium were replicated 6 times. The nutrient solutions are based on those used by Chouteau (6). The nitrate solution was made up as follows: KH9PO, (2 meq/l), MgSO4 (1.5 meq/l), Ca(NO3 ) 2 (5 meq/l). In both the ammonium and urea solutions, Ca ( NO3) 2 was replaced by an equivalent of CaSO4 (5 meq/l), and the nitrogen level was kept constant by the addition of the equivalents of (NH4)2SO4 or CO(NH2)2. Thus sulfate was the only variable. The micronutrients were supplied in all the 3 treatments as:

7 mation were also used for calcium and magnesium

in boiling water extracts. Total oxalate was determined according to Baker (3). Free oxalate, oxalate soluble in boiling water, was estimated but found only in trace amounts. Uronic acids also were determined using the manometric method of Tracey (21) in which CO2 evolved on decarboxvlation by 12 % (W/W) HCI is measured in a Van Slyke apparatus. The results were then calculated in meq per 100 g dry plant material by dividing the weight of 0O2 evolved by 44 assuming all the CO2 was derived from pectic substances. The alkalinity of the ash was determined by ashing 200 mg of dry plant material at 4800 for 24 hours. By means of a back titration using 0.1 N HCI followed by 0.1 N KOH, excess alkalinity could be determined. On the assumption that the inorganic anions other than nitrate are not affected by ashing, this valtue should be equivalent to the total organic anions in the dry plant material. Organic acids were determined on the fresh plant material. Using samples of 5 g of tissue, the organic acids were extracted by repeated treatments with boiling water after maceration of the plant tissue. The acids were estimated by partition chromatography using a silica gel column, after their isolation, as described in detail by DeKock and Morrison (10). The results of the analyses are expressed in terms of meq per 100 g dry weight for comparative purposes. Phosphorus is considered monovalent and sulfur divalent following the work of Dijkshoorn (12).

Fe-EDTA (2.80 jug Fe/ml), MnCl24H,O (0.550 JUg Mn/ml), H3B,03 (0.330 Jag B/ml), ZnCl, (0.065 jug Zn/ml), CuCl22H2O (0.064 ,ugC,u/ml), Na9MoO4

(0.046 ug Mo/ml). At the beginning of the experiment the pH of all the solutions was increased to 5.5 with dilute Ca(OH)2 solution. During the growth period the pH in all treatments was adjusted back to 5.5 every 2 or 3 days with Ca (O1H) 2 solution or 0.02 N H2SO4. After 14 days the nutrient solutions were completely renewed. During growth occasional qualitative checks were also made on the nitrate content in the solutions of the ammonium and urea series, and for the ammonium nitrogen content in the urea treatment. As these proved to be negative it is perhaps legitimate to assume that nitrogen was absorbed in the forms supplied. After 20 days growth in the nutrient media the plants were harvested and divided into leaves, petioles, stems, and roots and each replicate weighed. Similar tissues from each treatment were bulked together. Weighed samples, obtained as soon as possible after harvesting were stored in polythene bags at -15. The remaining weighed fresh material was dried at 850 to constant weight and ground to a fine powder using a micro hammer mill. Chemical estimations were made on both the fresh and dried plant material. The dried material was used for the estimation of total nitrogen, nitrate, inorganic sulfur, inorganic phosphorus, chloride, calcium, magnesium, potassium, sodium, oxalate, alkalinity of ash, and uronic acids. Total nitrogen, nitrate, inorganic sulfur, inorganic phosphorus and chloride were determined as described in another publication (15). Calcium, magnesium, potassium, and sodium were determined on 0.1 N hydrochloric acid extracts of the ash. Potassium and sodiuim were estimated by flame photometry, calcium as oxalate and magnesium by atomic absorption spectrometry. The same methods of esti-

Results
pH Changes in the Nutrient Soluitions During Growth. The well-known tendency for an increase
in pH in the external medium with nitrate nutrition and a corresponding decrease with ammonium nutrition was found (fig 1). Urea had an inter7.0
Sol ut ions

changed

pH
40

5 GROWTH

15 10 PERIOD (days)

20

FIG. 1. pH Changes in the nutrient media during growth (pH adjusted back to 5.5 after every determination). (A) NO3, (-) urea, ( 0 ) NH4.

IR

I'LANT PHYSIOLOGY

mediate effect, the nlutrieint me(liutm becoming aci(lic duiring the growth perio(l. Symitptomits at Harvest and Dry Hlaltter Yields. On harvesting the plants, marked(ldifferences betweeni the 3 treatments were observed. In comparison with the nitrate plants, the ammonitim fed plants were small with very (lark green leaves and] a very poorly developed sttibby root system. In contrast the nitrate plants were very well (levelope(I an(l had an extremely large root sy-stem. The tipper parts of the urea plants were intermediate in size althouigh the growth was rather spinidly, the leaves showing necrosis at the tips and a pale green colorverging on the appearance of nitrogen deficiency. Suirprisingly however, the roots were very well (levelope(l. The lower yields obtained in all tisstues from the ammoniulm plants in comparison with the corresponding tisstues of nitrate fe(d plants show out very clearly (table I). This has been observed l-, other
Table I. Influence in the Form of Nitrogen Nutrition on the Dry Matter Yields of Leaves, Petioles, Stemiis and Roots of Tomtato (g per 10 Plants)

Table II. Influienec of tli Form of Nitrogen Nutrition on thle Nitrogen Content of thle Leaz.es, Petioles, Stems and Rooiv isf TI Wato) (meq per 100 g drv wet)
(A mllOnlovalcnt)

N Souirce

NO,
Urea

NH4

Leaves Petioles Stems Roots 294 280 134 152 171 157 79 73 313 192 340 153

N Source NO.,

NH.1

ILeaves Petioles Stems Roots 9.9 12.6 10.2 30.2 6.5 9.7 3.4 15.3 4.8 2.8 53 9.6

N-orkers uising tomato (8, 9). Urea is intermedliate ini its effect except for the root weights which were almost equial to those of the nitrate treatment. As the root system of the uirea plants developed under a low pH regime in the nuttrieilt mediuim, it is therefore very unlikely that the low pH is solely responsible for the poor root development in the ammoniutm treatment.

Nitrogent Contentts. Muich lower contenits of nitrogen occtirred in the tissuies of the turea plants than in tissues from corresponding tissues from either of the other 2 treatments (table II). The somewhat higher concentrations of nitrogen in the ammonia plants as compared with the nitrate plants may be caused to some extent by concentrationl effects because of the lower yields in this treatment. Cation Com1zposition. The cation contents in variouts tissues of the tomato plant in relationi to the form of nitrogein nuitrition are shown in table III. The highest concentration of individual cations, other than soditum, occturred in the tissues of nitrate plants and was lowest in the ammoniutm fed plants. The same is also true for the total cation concentration. The well-known increase of potassiuim in stem and petiole tissues, and the reduction in calcium concentration from the leaf to the root was also apparent in all 3 treatments. The resutlts are generally in agreement with the findings of a ntumber of the other writers, from stuidies in nitrate and ammonium nultrition (1, 6, 13, 14, 19, 22). As dry matter yields were also lowest in the ammonitim plants the lower contents of cations in this treatment can not have resulte(d from concentratioin or diluition effects and muist, therefore, be dependent oIn differences in ion uiptake. Potassium and sodium occuir in plant tissuies almost totally in ionic form. \Vith calcitum anid( magnesiuim this is not the case. In order to obtain

Table III. Influence OJ tlh F )rm of N'itroqen Nutrition on Ihe Ino(ryanic Ca/ion Content in the Lcazc-es, Pe tiolet. . Stems, (ind Roits of T(cmato( ( meq ter 100 g dry zet)
C A T I 0 N S K Ca Mg (me(q per 100 g dry -\t) 161 58 30 27 34 134 25 29 62 176 126 38 110 30 162 90 61 17 162 86 35 127 82 31 18 54 50 93 44 40 40 27 105 43 11 38 Na
19

Total

Tisstue

N Soutrce

Leaves

NO.,
Urea

NH
Petioles

N\O.,
'Urea
NH1

Stellm.

NO.,

Urea

N O., NH14
Root
Urea

NH4

15 15 18 30 26 18 26 18 16 15 13

268 210 131 358 332 194 301 266 140 193 187 105

KIRKBY AND MENGEL-IONIC BALANCE IN RELATION TO NITROGEN NUTRITION9

a measuire of diffuisible cations, so that the difftusible cation-anion balance couild be considered, boiling water extracts were made on the dry plant material for calcium and magnesium estimation. The resuilts are shown in table IV. Althoutgh about 50 % of the total calcium was present in ionic form in the leaves, only about 10 % was water soltible in the petioles and 15 % in the stems and roots. A similar pattern is also true for magnesium except that a higher percentage than calcitum was found in ionic form in all tissues. Anion Composition. The inorganic anion contents in table V show that in the leaves and petioles the nitrate plants have the lowest inorganic anion concentration, and urea the highest. It would be expected that the inorganic content of nitrate fed plants shoutld be low as nitrate the major anion absorbed by the plant is converted largely to organic form. This is especially the case in the upper plant parts at the significant site of nitrate reTable IV. Influence of tile Form of Nitrogen Nutrition on the Content of Water Soluble Ca and M1g in the Leaves, Petioles, Stems, and Roots of Tomtato Results expressed as a percentage of total Ca and total Mg.
Tissue
Leaves
N Source
% Ca
% Mg

NO. Urea

NH4
Petioles
Stems
Roots

N03 Urea

NH4
NO3 Urea

NH4
Urea N,H

NO3

50 48 36 11 9 9 14 17 16 17 15 21

85 65 53 37 39 35 27 23 27 44 36 89

duiction in tomato (5). When ammonitum ions are taken up higher amounts of inorganic anions are also necessary to maintain ionic balance. As the diffusible cation concentration in the tissues of the ammonium plants is rather low, however, the somewhat lower concentrations of inorganic anions, than in the urea plants and the corresponding stems and root tisstues of the nitrate fed plants, may be explained. This effect can be seen from the restults of the inorganic anions expressed as a percentage of the diffusible cations where the urea and nitrate tissues have a lower percentage of their diffulsible cations bound with organic anions than corresponding ammonium tissues. The individual inorganic anions in the plant tissues investigated follow the same pattern in all 3 treatments, phosphate being highest in the roots, chloride in the stems and petioles and sulfate in the leaves. In most plant tissuies the prevailing pH is sutch that organic acids are present as the salts of inorganic cations. It is only in a few rather atypical plants that free acids are present to any marked extent. The non-volatile organic acids shown in table VI may thus be considered to contribute to the ionic equilibrium largely as organic anions. The influence of the form of nitrogen nutrition is shown very clearly, nitrate tissues having very much higher concentrations of organic acids than corresponding ammonitum tissues. This confirms the findings of Clark (8) and Coic (9) for tomato leaves and other workers using a nuimber of plant species (6,13, 14). From the literatuire there appears to be no earlier work on the effect of uirea nitrogen on the concentration or composition of the organic acid fraction in the plant. Our resuilts show the intermediate position of turea between nitrate and ammonium nutrition in this respect. There is also a marked effect on the concentration of the acids between tissuies of the same plant, the organic acids falling in concentration from the leaf to the root.

Table V. Infliuence of tile Form of Nitrogen XN'utrition on Content of Inorga,nic Ani ns in the Leaves, Petioles, Stems and Roots of T( mato (meq per 100 g dry zi't) INORGANIC ANIONS Total % of N Source Tissv:e C1H2PO4N03 SO4-Duf fusible cations (meqI per 100 g dry wt) 28 51 Lea, es 22 12 4 13 NO3 53 15 69 Urea 23 31 81 64 35 15 14 NH4 33 73 NO.. 16 12 15 30 Petioles 61 20 130 89 21 98 77 29 49 20 NH4 33 67 NO3 20 26 8 13 Stems 52 91 59 12 20 Urea 66 56 27 15 14 NH4 63 85 27 13 35 Roots 10 NO., 62 84 32 35 35 U-ea 72 53 17 21 15 NH4
.. .. ..

..

10

PLANT PHYSIOLOGY

Diffusible Cation-Anion Balantce. Figure 2 illustrates graphically the relationship between the diffusible cations and diffusible anions. The results show a very highly significant relationship (r = 0.992, b = 0.916+0.056, P <0.001), which infers that the most important diffusible cations and anions have been taken into account. Variations from the ideal 1:1 ratio may be accounted for by the cumulative errors of chemical analysis or omissions from the balance suich as proteins and organic bases. Non-Diffusible Cations and Anions. Table VII shows the comparison of the totals of bound calcium and magnesium against total oxalate and uronic acids. There is a remarkably good agreement between the cation and anion totals (r = 0.944, b = 1.1190.124, P<0.001). It must be considered, however, that althouigh oxalate was found to be present almost totally in salt form, this may not

be the case for the uironic aci(ds. Boiliig water also is not ideal for the extraction of diffuisible calcium and magnesium. Moreover, some non-diffusible magnesium is present in chlorophyll, although this is only a very small fraction of the total magnesium concentration (16). The close agreement between the results in table VII infers, however, that non-diffusible calciuim and magnesiuim ions are largely associated with pectate and oxalate. The form of nitrogen nutrition shows exactly the same effect with the oxalate content as with the diffusible non-volatile organic acids discuissed previously, highest concentrations being fouin(d in the tissues of nitrate fed plants. In agreement with other workers rather high levels of oxalate were fouind (8, 9). It is of interest to note that the tironic acid content was not so greatly influenced by the form of nitrogen nutritioni as the non-volatile organic acids. This is, as would be

Table VI. Influence of t11 Form of Nitrogen Nutrition on the Content of Non-Volatile Organic Acid Anions in the Lcazcs, Pctiolcs, Stems, and Roots of Tomato (mneq per kg fJ ret)
Tissue

N source

Fumaric
1.2 0.4 0.5 1.0 0.3

Leaxves
Petioles Stems

NO3 Urea NH4

NO3
Urea NH4 NO3 Urea NH4 NO3 Urea NH4

o.5
1.8 03 0.9 0.9 0.2 1.5

Roots

Succinic Malonic (meq per kg fr wt) 1.2 1.8 0.9 0.8 08 1.1 0.3 0.3 0.8 0.4 0.6 0.6 0.8 0.4 0.6 2.4 2.1 3.0 16 6.3 0.8

Malic
130.2 25.5 5.9 95.6 66 5 14.7 70.1 43.6 7.4

Citric
47.4 55.6 10.6

Total

13.7
9.1 1.2

11.5 4.8 1.1 2.5 2.7 1.1 9.3 3.0 0.3

181.8 83.2 18.9 108.4 71.9 17.$ 75.6 47.8 12.9 29.0 20.2 3.8

Table VII. Influence of tlhc Formn of Nitrogen Nutrition on the Contents of Bound Calciwnsl and Ilagncsium, and Oxalate and Uronic Acids in the Leaves, Petioles, Stems, and Roots of Tomtato (meq per 100 g dry ret) Cations

Tissuie
Leaves

Total

.Aiioils

N Source
NO3 Urea

Ca
81 69
40

Mg (meq per 100 g dry

5 10 86 79

w-t)

Oxalate Uronic Acids

41
25

Total

Petioles

NH4 NO3 Urea

NH4 NO3

44 40
46

85r

fi;

112 101 56
68 42
37 34 30

12 24
19 11

136 120 67
100 91 55

61 49 16
58 43 18 22
16

69 68 63
54

Stems

Urea
NH4

Roots

NO3

Urea
NH4

25 23 13 22 17 1

130 11, 79 112

50

59
51 31

_5

,3 ~13

Q3

~,s
42

11 KIRKBY AND 'MENGEL-IONIC BALANCE IN RELATION TO NITROGEN NUTRITION1

obtained by the other chemical estimations described earlier. The results are shown in figure 3. The very high significant relationship (r= 0.975, b = 0.997+ 0.071, P<0.001) between these 2 independent estimations of organic anions gives a very good indication that no anions or cations have been omitted from the balance in the results discussed previously. The results also demonstrate the usefulness of the alkalinity of the ash method in estimating organically bound anions. Total Cation-Anion Balance. The contribution of the individual fractions to the total cation-anion balance in the various plant tissues in relation to the form of nitrogen are shown in table VIII. The overall effect shows out very clearly in all tissues. A very close balance occurs in all treatments. Nitrate tisstues are highest in cations and anions, ammonium tissues lowest and those of urea are intermediate.

ANIONS(meq/100g) FIG. 2. The relationship between the diffusible cations (Ca++, Mg++, K+, Na+) and diffusible anions 5 , H2P04-, Cl- and non-volatile organic (NO3-, S04 acid n-iions) in meq per 100 dry wt. (A NO3, (N) urea, (0) NH4. y = 23.45 + 0.916 x.

100 DIFFUSIBLE

200

Discussion
The experimental data give very good stupport to the concept of a cation-anion balance in different plant tissues, maintained by the diffusible and indifftusible organic and inorganic, cations and anions which were determined. In each tisstue, independent of the form of nitrogen nutrition, the total cations are fairly well balanced by total anions. There is also a remarkably good agreement between the bound calcium and magnesium, and oxalic and uronic acids. It thus seems probable that these 2 organic constituents are mainly balanced by calcium and magnesium ions. The different forms of nitrogen ntutrition resulted in very different yields and growth habits of the plants. It is clear that there is a very close

expected, that the formation of primarv structures and cell walls of the plant are less dependent of the form of nitrogen nutrition. Total Organic Anions. The results discussed earlier indicate the very close relationship between the total cations and anions in all plant tissues. In order to confirm that all the anions and cations contributing to the balance had been taken into account, the total amounts of organic anions as obtained by the alkalinity of the ash method were graphed against the sum of the organic anions

Table VIII. Inzfluence of the Formii of Nitrogen Nutrition on the Cation-anion Balance in the Leaves, Pctioles, Stemns, and Roots of Tomato (meq fcr 100 g drv wt'
CATIONS
Tissue Leaves
Petioles

Non volatile

ANIONS Oxalate Total

N Source

Indiffusible
86 79
52

Diffusible

Organic
Total 268
210 131

NO3
Urea NH4

182
131 79
222 212 127

NO3 Urea
NH4

Stems

NO3 Urea

136 120 67 99 91
55

NH4
Roots
NO3 Urea

59
51

NH4

31

202 175 85 134 136 74

358 332 194 301 266 140 193 187 105

Inorganiic Uronic acids (meq per 100 g dry wt) 51 44 117 40 69 41 46 64 11 69 73 147 68 130 94 63 98 18 54 67 114 50 91 58 535 56 12 56 85 45 52 84 30 42 53

25
8 61 49 16

41

253
129

175

350 341 195

293

58
43 18

242

22
16 1

141 208 182 101

12

PLANT PHYSIOLOGY

A/
200
0-

4.

Al7

-E

.7s ;J-~~
lOO1

<I:

*/~~o

100
ORGANIC

200

ANIONS (meq /100g)

FIG. 3. The relationshlii) betsveen the alkalinity of the

itrate
organic

ash and the conitent of organic anions (total acild aniions and uronic acids) in me( per 100 drx-wt. (A) NO., (U) uirea, (* NHj. y 17.17 + 0.997 x

free

initeraction between the uiptake of ionls by the plant ariationis in vield may anld drv matter yield. therefore produce concentratioin or dilution effects onl catioIn anid anlioIn constituienits. These effects, however, shouil(d be similar for both catioils aind anioins. As the restilts showed, this was not the of yield differeinces case, so that the influiencc caninot accouint for constitueint changes in the cation-anion equilibrium brouight about by the 3 forms of ilitrogen nutritioin. \Vhein conisiderinig the questioin of cation-ainion balance in relationi to different nitrogen soturces it muist be kept in mind that in comparison with other nittrieints, the plant needs very high quaintities of nitrogeni in or(ler to realize its inherent reqtiiremenits. Thus with nitrate nultritioin the plant has to take tip high amouints of anions while with amiimoniutnm Inlutritioin high quLaintities of cationis must be absorbed. Already at this initial step with the uiptake of niitrate or ammoniulm ions into the cell, a halance has to occur otherwise the electropotential drop between the cell and medium wouldl be deleteriouis to the cell. The uiptake of nitrate has, therefore, to he accompainie(d by cations or by an exchainge of anions, juist as the uptake of ammoniulmln
iOlns muist be accompainied by anionls or the

supply. WN hether the process of the uiptake of nitrate or ammoniuim ions is an active process mediated by a carrier or a passive process along anl electro-chemical gradient is a secondary quiestion in this context. \WNhat is important is that electroneutrality mtust be maintainied both in the plant and the nuitrient medium. Nitrate taken tip from the nuitrienit soltution does not remain in ionic form. It is reduiced and (luring this reduiction the negative charge shifts from NO3to OH- which increases the pH of the system. - N3+H3 + 2H10 + OH(N5+03)811SH- + +8e This pH increase may lead to the accuimulation of organic acid anions either by the dissociationi of organic acids metabolically synthesized, as for example in the tricarboxylic acid cycle, or by the produiction of HCO0:- from CO2. As w,as demonstrated l)y Bedri et al (4) and Chouteau (7), the presence of bicarbonate in the nlultrient solutioin increases the organic acid content of the plant. It is feasible that a higher bicarbonate concentration in the tissue enhances bicarbonate incorporation into organic acids via CO,, fixation. Not all bicarbonate produced dulring niitrate re(Luction will remain in the tissuie, some will diffuise ouit of the root and increase the pH of the nuttrieint medliuim. This is in accordance with ouir observationls. The assimilation of nitrate therefore affects the cation-anion balance insofar as it provides a continuiouis source of negative charges which may be transferred to form organic aIniolns in the plant or retuirned to the nuttrienit mediuim as bicarbonate ions. The assimilation of stulphate ioIs shouldI also produice the same effect buit to a mulch less significant extent. \NVhether an organic anioin is formedl depenids oti whether or not the uiptake of the nitrate ion is accompaniied by the uiptake of a
100

e'
aCLa
D

80 -NH4
60

CL

U REA

20

0N

exchange of other cations from the root. From the resuilts showun in figuire 4 it is clear that the exchanige process mtist occuir. The excretion of hydroxyl or bicarbonate accouints for a greater anioin thain cation tuptake with nitrate nuitrition while hydrogen ioIn exchange resuilts from a higher catioin absorption with uirea and amlmoniumtl1 nitrogen

crLU
Lu

0c 20
C]<

40

fromii the niutrienlt media ( tptake in

FIG. 4. Differential remiioval of cationls anid aniionIs

meie by 10

plants).

KIRKBY AND LMENGEL-IONIC BALANCE IN RELATION TO NITROGEN NUTRITION

13

cation. If no cation is taken ulp the net result is the exchange of an equivalent amiount of nitrate from the nutrient solution for bicarbonate from the plant. The assimilation of ammonium ions leads to the production of hydrogen ions. NH4+ -* NH3 + H+ Thus with ammonium nutrition the pH of the tissue should be lowered. This assumption is in accordance with Mulder's observations (17) of lower pH in root and leaf tissues of peas supplied with ammonium rather than with nitrate nitrogen. Our own results show the same effect as can be seen in table IX. Differences are particuilarly
Table IX. Influtencc of this Form of Nitrogen Nutrition on the pH of Maccrated Tissues of Leaves, Petioles, Stems and Roots of Tomato Leaves
Petioles
Stems

Roots

NO3 Urea

NH1

5.50 5.5o 5 00

5.45 5.30 4.70

5.45 5.30 4.80

5.60 5.00 4.70

noticeable in the root tissues at the site of ammonium assimilation. As discussed above this lowered pH should reduce the accumulation of organic acid anions. This concept explains why with ammonium nutrition the content of organic anions is low and that of inorganic anions high. \Vith nitrate suipply the reverse is true. Hydrogen ions produced during ammonium assimilation will also diffuse out lowering the pH of the nutrient medium (fig 1) and also reduce cation uptake by competition effects (2). As urea is taken up as a molecule there is no need for it to be balanced by other ions. The total cations and organic anions should be intermediate between nitrate and ammonium nutrition. This is exactly what the results show (see tables III, V, VI). The assimilation of urea is assumed to be induced by urease (18) splitting urea into NH3 and CO. This process and the further incorporation of ammonia does not result in a change in pH. Thus the pH of the tissues of this treatment should lie between nitrate and ammonium tissues (table IX). In agreement with this result, the pH value of the nutrient solution is intermediate between the other 2 treatments as is also the content of organic acids (table VI). The urea plants had a lower total nitrogen content than the plants of the other 2 treatments. It is probable that in this case reduced growth in comparison with the nitrate plants was caused by an insufficient supply of nitrogen. From these results it is impossible to say whether the limiting factor was the uptake of urea or urease activity. In the case of ammonium nultrition the lowered pH of the tissue may have an important influence on growth processes. The pH values of macerated

plant tissues give only a rough indication of the real pH decrease which may occur at metabolic sites. It is possible for example that at low pH, photosynthetic CO2 fixation may be reduiced. The effect of the different nitrogen forms on the cation-anion balance is the same in leaves, petioles, stems and roots. Thus in addition to the result of Coic et al (9) for leaves, it is shown that the form of nitrogen nutrition exerts an overall effect on the plant. It is of interest that the upper parts of the plants contain higher concentrations of organic acids than the roots. Calcium shows a somewhat similar distribution. The high calcium content of the leaves relates to the uipward transport of calcium ions in the transpiration stream. During this transport, calcium ions muist be accompanied by equivalent amounts of anions. \V ith nitrate nutrition a rather high proportion of these anions will be nitrate. At the site of nitrate reduction which in the tomato occurs more especially in leaf tissue, organic anions may be accuimulated to balance calcium ions on the metabolic removal of the accompanying nitrate ions. The cation-anion ratios in all tissues were close to unity, so it must be assumed that the main components responsible for this equilibriuim have been taken into account. Other components in the plant with positive or negative charges do occuir and include phosphorylated compouinds, proteins, amino acids, hydrogen ions, and organic bases, but their contribution to the whole balance is low. A balanice between cations and anions in the plant infers that different cation species compete for the bulk of anions and vice versa. This is probably the reason why the increase in suipply of 1 ion species in the nuitrient medium decreases the uptake of a similarly charged ion species, where there is no specific competition for a carrier site.

Acknowledgments
The authors express their thanks to Professor T. XV. Walker, Department of Soil Science, Lincoln College, University of Canterbury. New Zealand, and to Dr. D. H. Jennings, Department of Botany, University of Leeds, England, for their interest in this +vork.

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